Summary of Work: Extensive genetic studies have established that the fidelities of replicative DNA polymerases and their associated proofreading 3'-exonucleases are the primary determinants of mutation rates. Structural information is now available for many of these enzymes, prompting structure-driven mutational analyses of fidelity. We have developed a bacteriophage system that permits the rapid analysis of the fidelities of mutant polymerases in vivo without a requirement for enzyme over-expression, purification, and fidelity analysis in vivo. Bacteriophage RB69 DNA polymerase supplied from a plasmid can replace the normal T4 DNA polymerase when the latter is mutationally inactivated, retaining high fidelity during T4 DNA replication. The structure of the RB69 DNA polymerase is described because it can crystallize, whereas the T4 DNA polymerase cannot. Using mutations that alter critical polymerase and exonuclease amino acids, we are characterizing the resulting changes in mutation rates using both reversion and forward-mutation tests in vivo and in vitro. We have altered a key residue in the active site to produce a strong mutator polymerase that preferentially produces transition mutations. We find that factors of increase caused by mutator mutations are higher when measured in vivo than in vivo. We also find that the combined mutator activity of a polymerase mutator and an exonuclease-defective mutator is not much greater than either alone. Clear differences are also seen between mutator specificities in vivo and in vivo.